Controlling fluorescent signal composition in optically-based nanopore sequencing

ABSTRACT

The invention is directed to optically based methods for nanopore sequencing of polynucleotides which comprise steps of (i) translocating a polynucleotide through a bore of a nanopore at a translocation speed, wherein nucleotides of the polynucleotide are labeled with fluorescent labels such that in free solution fluorescent labels of nucleotides are substantially quenched and wherein fluorescent labels within the bore are constrained such that substantially no detectable fluorescent signal is generated therein; (ii) exciting the fluorescent label of each nucleotide upon exiting the nanopore and prior to quenching with a preceding mutually quenching fluorescent label or a quenching agent; (iii) measuring a fluorescent signal generated by fluorescent labels exiting the nanopore, wherein the translocation speed is selected so that the measured fluorescent signal comprises fluorescence from substantially a single fluorescent label; and (iv) determining a nucleotide sequence of the polynucleotide from a sequence of measured fluorescent signals.

CROSS-REFERENCE TO RELATED APPLICATIONS AND SEQUENCE LISTING

This application claims benefit of priority to U.S. ProvisionalApplication No. 62/406,210, filed Oct. 10, 2016, which is incorporatedherewith in its entirety. A Sequence Listing is being provided inelectronic format. The Sequence Listing is provided as a file entitled02_QNTTPNZ02100_20220420 sequence listing ST25.txt, created Apr. 19,2022, which is 530 bytes in size. The information in the electronicformat of the Sequence Listing is incorporated herein by reference inits entirety.

BACKGROUND

Huber and colleagues discovered that by judicious selection of nanoporesfluorescent signals from fluorophore-labeled nucleotides of atranslocating polynucleotide may be rendered undetectable and that uponexit from the nanopore may regain detectability. This phenomena may beexploited practically in connection with another phenomena; namely, useof quenching agents or mutually quenching fluorescent labels on apolynucleotide, which in free solution quench one another. Applicationof these phenomena lead to a polynucleotide sequencing approach whereinnanopore-exiting nucleotides freed from nanopore-imposed constraints arecapable of excitation and emission of a detectable signal for aninterval just prior to quenching by a fluorescent label of a nucleotidethat preceded it through the nanopore. Huber et al, U.S. patentpublication 2016/0076091 and Huber, U.S. patent publication2016/0122812. The duration of the interval and translocation speed,among other factors, determine how many labels may contributefluorescence to measured signals of each signal collection cycle.

Optically based nanopore sequencing would be advanced and base callinggreatly simplified if these system parameters could be controlled sothat measured fluorescent signals of each signal collection cycleconsisted of fluorescence substantially from single labels.

SUMMARY OF THE INVENTION

The present invention is directed to methods, kits and systems foroptical detection and analysis of polymers, such as polynucleotides, inmicrofluidic and/or nanofluidic devices; in particular, the inventionincludes methods and systems using nanopores for determining nucleotidesequences of nucleic acids.

In some embodiments, the invention is directed to a method fordetermining a nucleotide sequence of a polynucleotide comprising thesteps of: (a) translocating a polynucleotide through a bore of ananopore at a translocation speed, wherein nucleotides of thepolynucleotide are labeled with fluorescent labels such that in freesolution fluorescent labels of nucleotides are substantially quenchedand wherein fluorescent labels within the bore are constrained such thatsubstantially no detectable fluorescent signal is generated therein; (b)exciting the fluorescent label of each nucleotide upon exiting thenanopore and prior to quenching with a preceding mutually quenchingfluorescent label or a quenching agent; (c) measuring a fluorescentsignal generated by fluorescent labels exiting the nanopore, wherein thetranslocation speed is selected so that the measured fluorescent signalcomprises fluorescence from substantially a single fluorescent label;and (d) determining a nucleotide sequence of the polynucleotide from asequence of measured fluorescent signals.

The present invention is exemplified in a number of implementations andapplications, some of which are summarized below and throughout thespecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate labeled polynucleotides translocating nanoporesat different speeds and the effect the different translocation speedshave on measured fluorescent signals.

FIGS. 2A-2C illustrate embodiments of the invention employing quenchingagents in a trans chamber, a cis chamber and in both cis and transchambers.

FIG. 3 illustrates an embodiment of the invention using a proteinnanopore and epi-illumination with a metal layer on the nanopore arrayto reduce background or TIR with FRET excitation.

FIG. 4 illustrates the basic components of a confocal epi-illuminationsystem.

FIG. 5 illustrates elements of a TIRF system for excitation of opticallabels in or near a nanopore array without FRET signal generation.

FIGS. 6A-6C illustrate embodiments employing two and three fluorescentlabels.

DETAILED DESCRIPTION OF THE INVENTION

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention. For example, particular nanoporetypes and numbers, particular labels, FRET pairs, detection schemes,fabrication approaches of the invention are shown for purposes ofillustration. It should be appreciated, however, that the disclosure isnot intended to be limiting in this respect, as other types ofnanopores, arrays of nanopores, and other fabrication technologies maybe utilized to implement various aspects of the systems discussedherein. Guidance for aspects of the invention is found in many availablereferences and treatises well known to those with ordinary skill in theart, including, for example, Cao, Nanostructures & Nanomaterials(Imperial College Press, 2004); Levinson, Principles of Lithography,Second Edition (SPIE Press, 2005); Doering and Nishi, Editors, Handbookof Semiconductor Manufacturing Technology, Second Edition (CRC Press,2007); Sawyer et al, Electrochemistry for Chemists, 2^(nd) edition(Wiley Interscience, 1995); Bard and Faulkner, Electrochemical Methods:Fundamentals and Applications, 2^(nd) edition (Wiley, 2000); Lakowicz,Principles of Fluorescence Spectroscopy, 3^(rd) edition (Springer,2006); Hermanson, Bioconjugate Techniques, Second Edition (AcademicPress, 2008); and the like, which relevant parts are hereby incorporatedby reference.

In one aspect, the invention relates to the use of nanopores,fluorescent quenching, and fluorescent signaling to sequentiallyidentify nucleotides of fluorescently labeled polynucleotide analytes.Such analysis of polynucleotide analytes may be carried out on singlepolynucleotides or on pluralities of polynucleotides in parallel at thesame time, for example, by using an array of nanopores. In someembodiments, nucleotides are labeled with fluorescent labels that arecapable of at least three states while attached to a polynucleotide: (i)A substantially quenched state wherein fluorescence of an attachedfluorescent label is quenched by a fluorescent label on an immediatelyadjacent monomer or by interaction with a quenching agent; for example,a fluorescent label attached to a polynucleotide in accordance with theinvention is substantially quenched when the labeled polynucleotide isfree in conventional aqueous solutions or buffers for studying andmanipulating the polynucleotide. (ii) A sterically constrained statewhile a labeled polynucleotide is translocating through a nanopore suchthat the free-solution movements or alignments of attached fluorescentlabels are disrupted or limited so that there is little or no detectablefluorescent signal generated from the fluorescent label. (iii) Atransition state wherein fluorescent labels attached to a polynucleotidetransition from the sterically constrained state to a quenched state asthe nucleotide of the fluorescent label exits the nanopore (during a“transition interval” or “interval”). In part, the invention is anapplication of the discovery that during the transition interval afluorescent label (on an otherwise substantially fully labeled andself-quenched or quenched polynucleotide) is capable of generating adetectable fluorescent signal and that the number of exiting labelscontributing to a measured signal may be (at least in part) controlledby controlling the translocation speed of the labeled polynucleotide. Iftranslocation speed (e.g. nucleotides exiting a nanopore per msec) ishigher than the transition rate (from signal-capable to quenched, i.e.the quenching rate), then measured fluorescent signals, or signalsamples, may contain contributions from more than one label. Thiscircumstance makes signal analysis more difficult and sequencedetermination possibly less accurate. In accordance with someembodiments, this problem may be ameliorated by adjusting translocationspeed, for example by reducing translocation speed, so thatsubstantially only one fluorescent label at a time contributesfluorescence to a measured fluorescent signal.

Without the intention of being limited by any theory underlying thisdiscovery, it is believed that the fluorescent signal generated duringthe transition interval is due to the presence of one or more freelyrotatable dipoles of the fluorescent labels that emerged from ananopore, which renders the fluorescent labels capable of generating afluorescent signal, for example, after direct excitation or viaexcitation via FRET. In some embodiments, the polynucleotide is a singlestranded polynucleotide, such as, DNA or RNA, but especially a singlestranded DNA. In some embodiments, the invention includes a method fordetermining a nucleotide sequence of a polynucleotide by recordingsignals generated by fluorescent labels as they exit a nanopore one at atime as a polynucleotide translocates through the nanopore. Atranslocation speed is selected to maximize the likelihood that measuredfluorescent signals comprise fluorescence from substantially only asingle label, wherein such selection may be made either by real-timeadjustment of parameters controllable during operation (such as thevoltage across the nanopores, temperature, or the like) or bypredetermined instrument set-up (e.g. reaction buffer viscosity, ionconcentration, or the like). Upon exit, each attached fluorescent labeltransitions during a transition interval from a constrained state in thenanopore to a quenched state on the polynucleotide in free solution.During the transition interval the label is capable of generating afluorescent signal which can be measured. In other words, in someembodiments, a step of the method of the invention comprises excitingeach fluorescent label as it is transitioning from a constrained statein the nanopore to a quenched state on the polymer in free solution. Asmentioned above, during this transition interval or period a fluorescentlabel is capable of emitting a detectable fluorescent signal indicativeof the nucleotide to which it is attached.

In some embodiments, “substantially quenched” as used above means afluorescent label generates a fluorescent signal at least thirty percentreduced from a signal generated under the same conditions, but withoutadjacent mutually quenching labels. In some embodiments, “substantiallyquenched” as used above means a fluorescent label generates afluorescent signal at least fifty percent reduced from a signalgenerated under the same conditions, but without adjacent mutuallyquenching labels.

The above concepts are illustrated in FIGS. 1A-1B, whichdiagrammatically show labeled polynucleotide (1000) translocatingthrough nanopore (1002). Labeled polynucleotide (1000) comprises twolabels “a” and “b” (for example, which may correspond to dC beinglabeled with “a” and dA, dG and dT being labeled with “b”, or the like).Labels of nucleotides free of nanopore (1002) are quenched, either byinteraction with other labels (1011) or by action of quenching agents(not shown). Labels of nucleotides inside of nanopore (1002) areconstrained and/or oriented (1014) so that they produce no detectablesignal during all or part of their transit through the nanopore. Asnucleotides of labeled polynucleotide (1000) emerge from exit (1015) ofnanopore (1002) they become capable of being excited by excitation beam(1010) and generating a detectable signal for an interval prior to beingquenched. If translocation speed V₁ is high then the distance (1008)traveled by a nucleotide prior to quenching may exceed theinter-nucleotide distance of polynucleotide (1000) so that more than onelabel (shown in FIG. 1A) contributes fluorescence to a fluorescentsignal collected by detector (1018), i.e. a measured fluorescent signal.If translocation speed V₂ is low then the distance (1008) traveled by anucleotide prior to quenching may approximately equal or be less thanthe inter-nucleotide distance of polynucleotide (1000) so that no morethan one label (shown in FIG. 1B) contributes fluorescence to afluorescent signal collected by detector (1018), i.e. a measuredfluorescent signal. Since the distance between adjacent labels is belowthe diffraction limit of excitation light (1010) no information isobtained about the ordering of the labels, although there are approachesto deduce such information using specialized algorithms, e.g. Andersonet al, U.S. provisional patent application 62/322,343; Timp et al,Biophys. J., 102: L37-L39 (2012); Carson et al, Nanotechnology, 26:074004 (2015). In the case of optical detection using fluorescent labelswith distinct emission bands, measured fluorescent signals may beseparated into two or more channels, e.g. using bandpass filters, inorder to assess the relative contributions of fluorescence from multiplelabels. However, as the number of fluorescent labels contributingfluorescence increases, e.g. 3, 4, or more, the difficulty indetermining a correct ordering of nucleotides increases. The signalintensities for two channels, e.g. corresponding to emission maxima oftwo fluorescent labels, is illustrated in FIG. 1A (1031 and 1032) wheretwo fluorescent labels contribute to a measured signal and in FIG. 1B(1041 and 1042) where a single fluorescent label contributes to ameasured signal. Intensity values represented by solid lines, e.g. 1033,are from label “a,” and intensity values represented by dashed lines,e.g. 1036, are from label “b”. The presence of solid and dashed lines inboth channels of FIG. 1A reflects overlapping emission bands of thefluorescent labels, which when collected together complicates analysisbecause amounts of a measured intensity are from both labels. In FIG.1B, where only a single fluorescent label contributes to the measuredsignal, intensity values do not contain contributions due to overlappingemission bands of other labels, thereby making label (and thereforenucleotide) determination easier.

Controlling Translocation Speed

The role of translocation speed of polynucleotides through nanopores andthe need for its control have been appreciated in the field of nanoporetechnology wherein changes in electric current are use to identifytranslocating analytes. A wide variety of methods have been used tocontrol translocation speed, which include both methods that can beadjusted in real-time without significant difficulty (e.g. voltagepotential across nanopores, temperature, and the like) and methods thatcan be adjusted during operation only with difficulty (reaction bufferviscosity, presence or absence of charged side chains in the bore of aprotein nanopore, ionic composition and concentration of the reactionbuffer, velocity-retarding groups attached or hybridized topolynucleotide analytes, molecular motors, and the like), e.g. Bates etal, Biophysical J., 84: 2366-2372 (2003); Carson et al, Nanotechnology,26(7): 074004 (2015); Yeh et al, Electrophoresis, 33(23): 58-65 (2012);Meller, J. Phys. Cond. Matter, 15: R581-R607 (2003); Luan et al,Nanoscale, 4(4): 1068-1077 (2012); Keyser, J. R. Soc. Interface, 8:1369-1378 (2011); and the like, which are incorporated herein byreference. In some embodiments, a step or steps are included for activecontrol of translocation speed while a method of the invention is beingimplemented, e.g. voltage potential, temperature, or the like; in otherembodiments, a step or steps are included that determine a translocationspeed that is not actively controlled or changed while a method of theinvention is being implemented, e.g. reaction buffer viscosity, ionicconcentration, and the like. In regard to the latter, in someembodiments, a translocation speed is selected by providing a reactionbuffer having a concentration of glycerol, or equivalent reagent, in therange of from 1 to 60 percent.

In regard to the former embodiments (with real-time translocation speedadjustment), a measure of whether one or more than one label iscontributing fluorescence to measured signals may be based on thedistribution of fluorescence intensity among a plurality of channelsover which fluorescence is collected. Typically the plurality ofchannels typically include 2, 3, or 4 channels corresponding to theemission bands of the fluorescent labels used, e.g. to label differentkinds of nucleotide. In a measured sample of fluorescence emanating froma region adjacent to a nanopore exit, if only a single label contributesto a measured signal, the relative distribution of signal intensityamong the different channels (e.g. 4 channels) could be representedideally as (1,0,0,0); (0,1,0,0); (0,0,1,0) or (0,0,0,1). On the otherhand, if more than one label contributed to a measured fluorescentsignal, the relative distributions would include non-zero values in morethan one channel, with a worse case being four different labelscontributing equally, which would appear as (0.25,0.25,0.25,0.25) in theabove representation. A measure which would vary monotonically between amaximum value corresponding to relative intensity distributions(1,0,0,0); (0,1,0,0); (0,0,1,0) or (0,0,0,1) and a minimum valuecorresponding to a relative intensity distribution of(0.25,0.25,0.25,0.25) may be used for controlling in real-time atranslocation speed. For example, an initial translocation speed couldbe lowered based on the value of such a measure that was near itsminimum. Such lowering may be implemented, for example, by lowering apotential voltage across the nanopores by a predetermined amount, afterwhich the measure could be re-calculated. Such steps could be repeateduntil the process was optimized, e.g. to maximize the fraction ofmeasurements wherein collected signals emanate from only a single label.

As mentioned above, translocation speeds depend in part on the voltagedifference (or electrical field strength) across a nanopore andconditions in the reaction mixture, or buffer, of a first chamber wherepolynucleotides are exposed to the nanopores (e.g. disposed in a solidphase membrane making up one wall of the first chamber). Polynucleotidecapture rates by nanopores depend on concentration of suchpolynucleotides. In some embodiments, conventional reaction mixtureconditions for nanopore sequencing may be employed with the invention(for controlling translocation speed by varying voltage potential acrossnanopores), for example, 1M KCl (or equivalent salt, such as NaCl, LiCl,or the like) and a pH buffering system (which, for example, ensures thatproteins being used, e.g. protein nanopores, nucleases, or the like, arenot denatured). In some embodiments, a pH buffering system may be usedto keep the pH substantially constant at a value in the range of 6.8 to8.8. In some embodiments, a voltage difference across the nanopores maybe in the range of from 70 to 200 mV. In other embodiments, a voltagedifference across the nanopores may be in the range of from 80 to 150mV. An appropriate voltage for operation may be selected usingconventional measurement techniques. Current (or voltage) across ananopore may readily be measured using commercially availableinstruments. A voltage difference may be selected so that translocationspeed is within a desired range. In some embodiments, a range oftranslocation speeds comprises those speeds less than 1000 nucleotidesper second. In other embodiments, a range of translocation speeds isfrom 10 to 800 nucleotides per second; in other embodiments, a range oftranslocation speeds is from 10 to 600 nucleotides per second; in otherembodiments, a range of translocation speeds is from 200 to 800nucleotides per second; in other embodiments, a range of translocationspeeds is from 200 to 500 nucleotides per second. Likewise, otherfactors affecting translocation speed, e.g. temperature, viscosity, ionconcentration, charged side chains in the bore of a protein nanopore,and the like, may be selected to obtain translocation speeds in theranges cited above.

In some embodiments, a device for implementing the above methods forsingle stranded nucleic acids typically includes providing a set ofelectrodes for establishing an electric field across the nanopores(which may comprise an array). Single stranded nucleic acids are exposedto nanopores by placing them in an electrolyte (i.e. reaction buffer) ina first chamber, which is configured as the “cis” side of the layeredmembrane by placement of a negative electrode in the chamber. Uponapplication of an electric field, the negatively single stranded nucleicacids are captured by nanopores and translocated to a second chamber onthe other side of the layered membrane, which is configured as the“trans” side of membrane by placement of a positive electrode in thechamber. As mentioned above, the speed of translocation depends in parton the ionic strength of the electrolytes in the first and secondchambers and the applied voltage across the nanopores. In opticallybased detection, a translocation speed may be selected by preliminarycalibration measurements, for example, using predetermined standards oflabeled single stranded nucleic acids that generate signals at differentexpected rates per nanopore for different voltages. Thus, for DNAsequencing applications, an initial translocation speed may be selectedbased on the signal rates from such calibration measurements, as well asthe measure based on relative signal intensity distribution discussedabove. Consequently, from such measurements a voltage may be selectedthat permits, or maximizes, reliable nucleotide identifications, forexample, over an array of nanopores. In some embodiments, suchcalibrations may be made using nucleic acids from the sample oftemplates being analyzed (instead of, or in addition to, predeterminedstandard sequences). In some embodiments, such calibrations may becarried out in real time during a sequencing run and the applied voltagemay be modified in real time based on such measurements, for example, tomaximize the acquisition of nucleotide-specific signals.

Embodiments Employing Mutually and Self-Quenching Labels

As mentioned above, in some embodiments, self- and mutually quenchingfluorescent labels may be used in addition to quenching agents in orderto reduce fluorescent emissions outside of those from labels onnucleotides exiting nanopores. Use of such fluorescent labels isdisclosed in U.S. patent publication 2016/0122812, which is incorporatedby reference. In some embodiments, monomers are labeled with fluorescentlabels that are capable of at least three states while attached to atarget polynucleotide: (i) A substantially quenched state whereinfluorescence of an attached fluorescent label is quenched by afluorescent label on an immediately adjacent monomer; for example, afluorescent label attached to a polynucleotide in accordance with theinvention is substantially quenched when the labeled polynucleotide isfree in conventional aqueous solution for studying and manipulating thepolynucleotide. (ii) A sterically constrained state wherein a labeledpolynucleotide is translocating through a nanopore such that thefree-solution movements or alignments of an attached fluorescent labelis disrupted or limited so that there is little or no detectablefluorescent signal generated from the fluorescent label. (iii) Atransition state wherein a fluorescent label attached to apolynucleotide transitions from the sterically constrained state to thequenched state as the fluorescent label exits the nanopore (during a“transition interval”) while the polynucleotide translocates through thenanopore.

In part, this example is an application of the discovery that during thetransition interval a fluorescent label (on an otherwise substantiallyfully labeled and self-quenched polynucleotide) is capable of generatinga detectable fluorescent signal. Without the intention of being limitedby any theory underlying this discovery, it is believed that thefluorescent signal generated during the transition interval is due tothe presence of a freely rotatable dipole in the fluorescent labelemerging from the nanopore, which renders the fluorescent labeltemporarily capable of generating a fluorescent signal, for example,after direct excitation or via FRET. In both the sterically constrainedstate as well as the quenched state, the dipoles are limited in theirrotational freedom thereby reducing or limiting the number of emittedphotons. In some embodiments, the polynucleotide is a polynucleotide,usually a single stranded polynucleotide, such as, DNA or RNA, butespecially single stranded DNA. In some embodiments, the inventionincludes a method for determining a nucleotide sequence of apolynucleotide by recording signals generated by attached fluorescentlabels as they exit a nanopore one at a time as a polynucleotidetranslocates through the nanopore. Upon exit, each attached fluorescentlabel transitions during a transition interval from a constrained statein the nanopore to a quenched state on the polynucleotide in freesolution. In other words, in some embodiments, a step of the method ofthe invention comprises exciting each fluorescent label as it istransitioning from a constrained state in the nanopore to a quenchedstate on the polynucleotide in free solution. As mentioned above, duringthis transition interval or period the fluorescent label is capable ofemitting a detectable fluorescent signal indicative of the nucleotide itis attached to.

In some embodiments, the invention includes an application of thediscovery that fluorescent labels and nanopores may be selected so thatduring translocation of a polynucleotide through a nanopore fluorescentlabels attached to monomers are forced into a constrained state in whichthey are incapable (or substantially incapable) of producing adetectable fluorescent signal. In some embodiments, nanopores areselected that have a bore, or lumen, with a diameter in the range offrom 1 to 4 nm; in other embodiments, nanopores are selected that have abore or lumen with a diameter in the range of from 2 to 3 nm. In someembodiments, such bore diameters are provided by a protein nanopore. Insome embodiments, such nanopores are used to force fluorescent labelsinto a constrained state in accordance with the invention, so thatwhenever a fluorescent label exits a nanopore, it transitions from beingsubstantially incapable of generating a fluorescent signal to beingdetectable and identifiable by a fluorescent signal it can be induced toemit. Thus, fluorescent labels attached to each of a sequence ofmonomers of a polynucleotide may be detected in sequence as theysuddenly generate a fluorescent signal in a region immediately adjacentto a nanopore exit (a “transition zone” or “transition volume” or“detection zone”). In some embodiments, organic fluorescent dyes areused as fluorescent labels with nanopores of the above diameters. Insome embodiments, at least one such organic fluorescent dye is selectedfrom the set consisting of xanthene dyes, rhodamine dyes and cyaninedyes. Some embodiments for determining a monomer sequence of apolynucleotide may be carried out with the following steps: (a)translocating a polynucleotide through a nanopore, wherein monomers ofthe polynucleotide are labeled with fluorescent labels wherein thenanopore constrains fluorescent labels within its bore into aconstrained state such that substantially no detectable fluorescentsignal is generated therein; (b) exciting the fluorescent label of eachmonomer upon exiting the nanopore; (c) measuring a fluorescent signal ina detection zone generated by the exiting fluorescent label to identifythe monomer to which the fluorescent label is attached; (d) quenchingfluorescent signals from excited fluorescent labels outside of thedetection zone, and (d) determining a monomer sequence of thepolynucleotide from a sequence of fluorescent signals. In furtherembodiments, fluorescent labels are acceptors of a FRET pair and one ormore donors of the FRET pair are attached to the nanopore within a FRETdistance of the exit.

In some embodiments, “substantially quenched” as used above means afluorescent label generates a fluorescent signal at least thirty percentreduced from a signal generated under the same conditions, but withoutadjacent mutually quenching labels. In some embodiments, “substantiallyquenched” as used above means a fluorescent label generates afluorescent signal at least fifty percent reduced from a signalgenerated under the same conditions, but without adjacent mutuallyquenching labels.

In some embodiments, a nucleotide sequence of a target polynucleotide isdetermined by carrying out four separate reactions in which copies ofthe target polynucleotide have each of its four different kinds ofnucleotide (A, C, G and T) labeled with a single fluorescent label. In avariant of such embodiments, a nucleotide sequence of a targetpolynucleotide is determined by carrying out four separate reactions inwhich copies of the target polynucleotide have each of its fourdifferent kinds of nucleotide (A. C, G and T) labeled with onefluorescent label while at the same time the other nucleotides on thesame target polynucleotide are labeled with a second fluorescent label.For example, if a first fluorescent label is attached to A's of thetarget polynucleotide in a first reaction, then a second fluorescentlabel is attached to C's, G's and T's (i.e. to the “not-A” nucleotides)of the target polynucleotides in the first reaction. Likewise, incontinuance of the example, in a second reaction, the first label isattached to C's of the target polynucleotide and the second fluorescentlabel is attached to A's, G's and T's (i.e. to the “not-C” nucleotides)of the target polynucleotide. And so on, for nucleotides G and T.

The same labeling scheme may be expressed in terms of conventionalterminology for subsets of nucleotide types; thus, in the above example,in a first reaction, a first fluorescent label is attached to A's and asecond fluorescent label is attached to B's; in a second reaction, afirst fluorescent label is attached to C's and a second fluorescentlabel is attached to D's; in a third reaction, a first fluorescent labelis attached to G's and a second fluorescent label is attached to H's;and in a fourth reaction, a first fluorescent label is attached to T'sand a second fluorescent label is attached to V's.

In some embodiments, a polymer, such as a polynucleotide or peptide, maybe labeled with a single fluorescent label attached to a single kind ofmonomer, for example, every T (or substantially every T) of apolynucleotide is labeled with a fluorescent label, e.g. a cyanine dye.In such embodiments, a collection, or sequence, of fluorescent signalsfrom the polynucleotide may form a signature or fingerprint for theparticular polynucleotide. In some such embodiments, such fingerprintsmay or may not provide enough information for a sequence of monomers tobe determined.

In some embodiments, a feature of the invention is the labeling ofsubstantially all monomers of a polynucleotide analyte with fluorescentdyes or labels that are members of a mutually quenching set. The use ofthe term “substantially all” in reference to labeling polynucleotideanalytes is to acknowledge that chemical and enzymatic labelingtechniques are typically less than 100 percent efficient. In someembodiments, “substantially all” means at least 80 percent of allmonomer have fluorescent labels attached. In other embodiments,“substantially all” means at least 90 percent of all monomer havefluorescent labels attached. In other embodiments, “substantially all”means at least 95 percent of all monomer have fluorescent labelsattached. Mutually quenching sets of fluorescent dyes have the followingproperties: (i) each member quenches fluorescence of every member (forexample, by FRET or by static or contact mechanisms), and (ii) eachmember generates a distinct fluorescent signal when excited and when ina non-quenched state. That is, if a mutually quenching set consists oftwo dyes, D1 and D2, then (i) D1 is self-quenched (e.g. by contactquenching with another D1 molecule) and it is quenched by D2 (e.g. bycontact quenching) and (ii) D2 is self-quenched (e.g. by contactquenching with another D2 molecule) and it is quenched by D1 (e.g. bycontact quenching). Guidance for selecting fluorescent dyes or labelsfor mutually quenching sets may be found in the following references,which are incorporated herein by reference: Johansson, Methods inMolecular Biology, 335: 17-29 (2006); Marras et al, Nucleic AcidsResearch, 30: e122 (2002); and the like. In some embodiments, members ofa mutually quenching set comprise organic fluorescent dyes thatcomponents or moieties capable of stacking interactions, such asaromatic ring structures. Exemplary mutually quenching sets offluorescent dyes, or labels, may be selected from rhodamine dyes,fluorescein dyes and cyanine dyes. In one embodiment, a mutuallyquenching set may comprise the rhodamine dye, TAMRA, and the fluoresceindye, FAM. In another embodiment, mutually quenching sets of fluorescentdyes may be formed by selecting two or more dyes from the groupconsisting of Oregon Green 488, Fluorescein-EX, fluoresceinisothiocyanate, Rhodamine Red-X, Lissamine rhodamine B, Calcein,Fluorescein, Rhodamine, one or more BODIPY dyes, Texas Red, Oregon Green514, and one or more Alexa Fluors. Representative BODIPY dyes includeBODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 581/591, BODIPY TR, BODIPY630/650 and BODIPY 650/665. Representative Alexa Fluors include AlexaFluor 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633,635, 647, 660, 680, 700, 750 and 790.

As above, in some embodiments, a monomer sequence of a targetpolynucleotide is determined by carrying out separate reactions (one foreach kind of monomer) in which copies of the target polynucleotide haveeach different kind of monomer labeled with a mutually- orself-quenching fluorescent label. In other embodiments, a monomersequence of a target polynucleotide is determined by carrying outseparate reactions (one for each kind of monomer) in which copies of thetarget polynucleotide have each different kind of monomer labeled with adifferent mutually quenching fluorescent label selected from the samemutually quenching set. In embodiments in which a mutually quenching setcontains only two dyes, then a selected monomer (say, monomer X) islabeled with a first mutually quenching dye and every other kind ofmonomer (i.e., not-monomer X) is labeled with a second mutuallyquenching dye from the same set. Thus, steps of the embodiment generatea sequence of two different fluorescent signals, one indicating monomerX and another indicating not-monomer X.

In some embodiments, a single fluorescent label (for example, attachedto a single kind of monomer in a polynucleotide comprising multiplekinds of monomers) may be used that is self-quenching when attached toadjacent monomers (of the same kind) on a polynucleotide, such asadjacent nucleotides of a polynucleotide. Exemplary self-quenchingfluorescent labels include, but are not limited to, Oregon Green 488,fluorescein-EX, FITC, Rhodamine Red-X, Lissamine rhodamine B, calcein,fluorescein, rhodamine, BODIPYS, and Texas Red, e.g. which are disclosedin Molecular Probes Handbook, 11th Edition (2010).

Embodiments Employing Quenching Agents

FIGS. 2A-2C illustrate different embodiments of the inventioncorresponding to where quenching agents are applied in a nanoporedevice: trans chamber only (FIG. 2A), cis chamber only (FIG. 2B), orboth cis and trans chambers (FIG. 2C). In FIG. 2A, labeledpolynucleotide (200) is illustrated translocating nanopore (206) ofsolid phase membrane (208) from cis chamber (202) to trans chamber(204). Immersed in trans chamber (204) are non-fluorescent quenchingagents (205) designated by “Q”. Quenching agents of the invention aresoluble under translocation conditions for labeled polynucleotide (200),and under the same conditions, quenching agents bind to single strandedpolynucleotides, such as (200), without substantial sequencespecificity. As explained more fully below, a large variety ofnon-fluorescent quenching agents are available for use with theinvention, which include derivatives of many well-known organic dyes,such as asymmetric cyanine dyes, as well as conjugates of such compoundsand oligonucleotides and/or analogs thereof. In this embodiment,selection of the type and concentration of quenching agent and thetranslocation speed define detection zone (210). In some embodiments,“detection zone” means a region or volume (which may be contiguous ornon-contiguous) from which fluorescent signals are collected to form theraw data from which information, such as sequence information, about alabeled polynucleotide is determined. Fluorescent labels in transchamber (204) outside of detection zone (210) are substantially quenchedby quenching agents (205) bound to the portion of labeled polynucleotide(200) in trans chamber (204). In some embodiments, quenching agentscomprise an oligonucleotide or analog conjugated to one or morequenching moieties based on organic dyes as described more fully below.Embodiments of FIG. 2A may be employed when, for example, solid phasemembrane (208) is or comprises an opaque layer so that fluorescentlabels in cis chamber (202) are substantially non-excited.

FIG. 2B shows substantially the same elements as those in FIG. 2A withthe exception that quenching agents (205) are disposed in cis chamber(202). This configuration may be desirable under circumstances whereundesired evanescent waves, or like non-radiative light energy, extendto cis chamber (202) and excite fluorescent labels which generatefluorescent signals that are collected. Quenching agents (205) that bindto labeled polynucleotide (200) in cis chamber (202) reduce or eliminatesuch fluorescent signals. In some embodiments, quenching agents (205)and cross-section of nanopore (206) are selected so that quenchingagents (205) are excluded from translocating through nanopore (206). Insome embodiments, this may be achieved by using protein nanoporeα-hemolysin and quenching agents comprising conjugates ofoligonucleotides or analogs thereof and one or more quenching compounds,as described more fully below.

FIG. 2C illustrates an embodiment where quenching agents (205) arepresent in both cis chamber (202) and trans chamber (204), whichprovides the advantages described for the embodiments of both FIGS. 2Aand 2B.

FIG. 3 illustrates an embodiment which includes the following elements:protein nanopore (300) disposed in lipid bilayer (302); epi-illuminationof fluorescent labels with opaque layer (308) in solid phase membrane(306) to prevent or reduce background fluorescence; and quenching agents(310) disposed in trans chamber (326). As above, polynucleotide (320)with fluorescently labeled nucleotides (labels being indicated by “f”,as with (322)) is translocated through nanopore (300) from cis chamber(324) to trans chamber (326). Oligonucleotide quenchers (310) aredisposed in trans chamber (326) under conditions (e.g. concentration,temperature, salt concentration, and the like) that permitshybridization of oligonucleotide quenchers (328) to portions ofpolynucleotide (320) emerging from nanopore (300). Nanopore (300) may beselected so that signals from fluorescent labels are suppressed duringtransit of the nanopore as described in Huber et al, U.S. patentpublication US 2016/0076091, which is incorporated herein by reference.Thus, when labeled nucleotides emerge from nanopore (300) in region(328) they become unsuppressed and capable of generating a signal. Withmost if not all forms of direct illumination (e.g. non-FRET) suchemerged labels would continue to emit fluorescence as they travelfurther into trans chamber (326), thereby contributing greatly to acollected signal. With quenching agents in trans chamber (326) that bindto the emerging polynucleotide, such emissions can be significantlyreduced and can define detection zone (328) from which collected signalscan be analyzed to give nucleotide sequence information aboutpolynucleotide (320). In some embodiments, a fluorescent signal from asingle fluorescent label is detected from detection zone (328) during adetection period as the labeled polynucleotide moves through thedetection zone. In other embodiments, a plurality of fluorescent signalsis collected from a plurality of fluorescent labels in detection zone(328) during a predetermined time period. In some embodiments, suchdetection period is less than 1 msec, or less than 0.1 msec, or lessthan 0.01 msec. In some embodiments, such detection period is at least0.01 msec, or at least 0.1 msec, or at least 0.5 msec.

Quenching agents of the invention comprise any compound (or set ofcompounds) that under nanopore sequencing conditions is (i)substantially non-fluorescent, (ii) binds to single stranded nucleicacids, particularly single stranded DNA, and (iii) absorbs excitationenergy from other molecules non-radiatively and releases itnon-radiatively. In some embodiments, quenching agents further bindnon-covalently to single stranded DNA. A large variety of quenchingcompounds are available for use with the invention including, but notlimited to, non-fluorescent derivatives of common synthetic dyes such ascyanine and xanthene dyes, as described more fully below. Guidance inselecting quenching compounds may be found in U.S. Pat. Nos. 6,323,337;6,750,024 and like references, which are incorporated herein byreference.

In some embodiments, a quenching agent may be a single stranded DNAbinding dye that has been covalently modified with a heavy atom that isknown to quench fluorescence (such as bromine or iodine), or covalentlymodified with other groups known to quench fluorescence, such as a nitrogroup or a azo group. An example of dye that is known to bind singlestranded DNA is Sybr Green (Zipper et al, (2004), Nucleic AcidsResearch. 32 (12)). Incorporation of a nitro, bromine, iodine, and/orazo groups into the cyanine Sybr Green structure provides a singlestranded DNA binding group moiety that will quench fluorescent labelsthat might be present on a DNA.

In some embodiments, quenching agents comprise a binding moiety and oneor more quenching moieties. Binding moieties may include any compoundthat binds to single stranded nucleic acids without substantial sequencespecificity. Binding moieties may comprise peptides or oligonucleotidesor analogs of either having modified linkages and/or monomers.Oligonucleotides and their analogs may provide binding topolynucleotides via duplex formation or via non-base paired aptamericbinding. In some embodiments, binding moieties comprise anoligonucleotide or analog thereof having a length in the range of from 6to 60 nucleotides. Such oligonucleotides or analogs may be conjugated toone quenching moiety or to a plurality of quenching moieties. In someembodiments, the plurality of quenching moieties conjugated to eacholigonucleotide or analog is 2 or 3. Quenching moieties conjugated to abinding moiety may be the same or different. In some embodiments,whenever a binding moiety is an oligonucleotide or analog, two quenchingmoieties are conjugated thereto, one at a 5′ end and one at a 3′ end ofthe oligonucleotide. Oligonucleotides or analogs having from 2 to 3quenching moieties may be synthesized using conventional linkage andsynthetic chemistries, for example, as disclosed in the references citedherein.

Oligonucleotides or analogs may be provided as a single species or theymay be provided as mixtures of a plurality of oligonucleotides oranalogs with different sequences, and therefore, different bindingspecificities. In some embodiments, oligonucleotides or analogs arerandom sequence polymers; that is, they are provided as mixtures ofevery possible sequence of a given length. For example, sucholigonucleotides or analogs may be represented by the formulas, “NNNNNN”for 6-mers, or “NNNNNNNN” for 8-mers, wherein N may be A, C, G or T, oran analog thereof.

“Analogs” in reference to oligonucleotides means an oligonucleotide thatcontains one or more nucleotide analogs. As described in the definitionsection, a “nucleotide analog” is a nucleotide that may have a modifiedlinkage moiety, sugar moiety or base moiety. Exemplary oligonucleotideanalogs that may be used with the invention include, but are not limitedto, peptide nucleic acids (PNAs), locked nucleic acids(LNAs)(2′-O-methyl RNA), phosphorothioate oligonucleotides, bridgednucleic acids (BNAs), or the like.

In some embodiments, oligonucleotide binding moieties comprise universalbases; that is, they contain one or more nucleotide analogs that canreplace any of the four natural nucleotides without destabilizingbase-pair interactions. Nucleotide analogs having universal baseproperties are described in Loakes, Nucleic Acids Research, 29(12):2437-2447 (2001), which is incorporated herein by reference. In someembodiments, oligonucleotide binding moieties comprise 2′-deoxyinosine,7-deaza-2′-deoxyinosine, 2-aza-2′-deoxyinosine, 3-nitropyrrolenucleotides, 5-nitroindole nucleotides, or the like.

In some embodiments, quenching agents may comprise a combination of twoor more compounds that act together to quench undesired fluorescentsignals of a single stranded labeled polynucleotide. For example, aquenching agent may comprise an oligonucleotide (e.g., polydeoxyinosine)that may form a duplex with the labeled polynucleotide and separately adouble stranded intercalator that is a quencher. Thus, whenever thepolydeoxyinosine binds to a labeled polynucleotide, the quenchingintercalator binds to the resulting duplex and quenches fluorescentsignals from the polynucleotide.

Any synthetic dye that can detectably quench fluorescent signals of thefluorescent labels of a labeled polynucleotide is an acceptablequenching moiety for the purposes of the invention. Specifically, asused in the invention, the quenching moieties possess an absorption bandthat exhibits at least some spectral overlap with an emission band ofthe fluorescent labels on a labeled polynucleotide. This overlap mayoccur with emission of the fluorescent label (donor) occurring at alower or even higher wavelength emission maximum than the maximalabsorbance wavelength of the quenching moiety (acceptor), provided thatsufficient spectral overlap exists. Energy transfer may also occurthrough transfer of emission of the donor to higher electronic states ofthe acceptor. One of ordinary skill in the art determines the utility ofa given quenching moiety by examination of that dye's excitation bandswith respect to the emission spectrum of the fluorescent labels beingused.

Typically, fluorescence quenching in the invention occurs throughFluorescence Resonance Energy Transfer (FRET or through the formation ofcharge transfer complexes) between a fluorescent label and a quenchingmoiety of the invention. The spectral and electronic properties of thedonor and acceptor compounds have a strong effect on the degree ofenergy transfer observed, as does the separation distance between thefluorescent labels on the labeled polynucleotide and the quenchingmoiety. As the separation distance increases, the degree of fluorescencequenching decreases.

A quenching moiety may be optionally fluorescent, provided that themaximal emission wavelength of the dye is well separated from themaximal emission wavelength of the fluorescent labels when bound tolabeled polynucleotides. Preferably, however, the quenching moiety isonly dimly fluorescent, or is substantially non-fluorescent, whencovalently conjugated to a oligonucleotide or analog. Substantiallynon-fluorescent, as used herein, indicates that the fluorescenceefficiency of the quenching moiety in an assay solution as described forany of the methods herein is less than or equal to 5 percent, preferablyless than or equal to 1 percent. In other embodiments, the covalentlybound quenching moiety exhibits a quantum yield of less than about 0.1,more preferably less than about 0.01. In some embodiments, thefluorescence of fluorescent labels associated with a quenchingoligonucleotide of the invention is quenched more than 50% relative tothe same oligonucleotide associated with the same fluorescent labels inthe absence of the covalently bound quenching moiety. In anotherembodiment, the fluorescent labels are quenched more than 90% relativeto the unlabeled oligonucleotide. In yet another embodiment, the nucleicacid stains are quenched more than 95% relative to the unlabeledoligonucleotide.

In some embodiments, a quenching moiety may be a pyrene, an anthracene,a naphthalene, an acridine, a stilbene, an indole or benzoindole, anoxazole or benzoxazole, a thiazole or benzothiazole, a4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine, a carbocyanine,a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, aperylene, a pyridine, a quinoline, a coumarin (includinghydroxycoumarins and aminocoumarins and fluorinated and sulfonatedderivatives thereof (as described in U.S. Pat. No. 5,830,912 to Gee etal. (1998) and U.S. Pat. No. 5,696,157 to Wang et al. (1997),incorporated by reference), a polyazaindacene (e.g. U.S. Pat. No.4,774,339 to Haugland, et al. (1988); U.S. Pat. No. 5,187,288 to Kang,et al. (1993); U.S. Pat. No. 5,248,782 to Haugland, et al. (1993); U.S.Pat. No. 5,274,113 to Kang, et al. (1993); U.S. Pat. No. 5,433,896 toKang, et al. (1995); U.S. Pat. No. 6,005,113 to Wu et al. (1999), allincorporated by reference), a xanthene, an oxazine or a benzoxazine, acarbazine (U.S. Pat. No. 4,810,636 to Corey (1989), incorporated byreference), or a phenalenone or benzphenalenone (U.S. Pat. No. 4,812,409Babb et al. (1989), incorporated by reference).

In other embodiments, quenching moieties that are substantiallynon-fluorescent dyes include in particular azo dyes (such as DABCYL orDABSYL dyes and their structural analogs), triarylmethane dyes such asmalachite green or phenol red, 4′,5z-diether substituted fluoresceins(U.S. Pat. No. 4,318,846 (1982)), or asymmetric cyanine dye quenchers(PCT Int. App. WO 99 37,717 (1999)).

In embodiments where the quenching moiety is a xanthene, the syntheticdye is optionally a fluorescein, a rhodol (U.S. Pat. No. 5,227,487 toHaugland, et al. (1993), incorporated by reference), or a rhodamine. Asused herein, fluorescein includes benzo- or dibenzofluoresceins,seminaphthofluoresceins, or naphthofluoresceins. Similarly, as usedherein rhodol includes seminaphthorhodafluors (U.S. Pat. No. 4,945,171to Haugland, et al. (1990), incorporated by reference). Xanthenesinclude fluorinated derivatives of xanthene dyes (Int. Publ. No. WO97/39064, Molecular Probes, Inc. (1997), incorporated by reference), andsulfonated derivatives of xanthene dyes (Int. Publ. No. WO 99/15517,Molecular Probes, Inc. (1999), incorporated by reference). As usedherein, oxazines include resorufms, aminooxazinones, diaminooxazines,and their benzo-substituted analogs.

In further embodiments, the quenching moiety is an substantiallynonfluorescent derivative of 3- and/or 6-amino xanthene that issubstituted at one or more amino nitrogen atoms by an aromatic orheteroaromatic ring system, e.g. as described in U.S. Pat. No.6,399,392, which is incorporated herein by reference. These quenchingdyes typically have absorption maxima above 530 nm, have little or noobservable fluorescence and efficiently quench a broad spectrum ofluminescent emission, such as is emitted by chemilumiphores, phosphors,or fluorophores. In one embodiment, the quenching dye is a substitutedrhodamine. In another embodiment, the quenching compound is asubstituted rhodol.

In still other embodiments, a quenching moiety may comprise one or morenon-fluorescent quenchers known as Black Hole Quenchers™ compounds(BHQs) described in the following patents, which are incorporated hereinby reference: U.S. Pat. Nos. 7,019,129; 7,109,312; 7,582,432; 8,410,025;8,440,399; 8,633,307; 8,946,404; 9,018,369; or 9,139,610.

Additional quenching moieties are disclosed in the following, which areincorporated herein by reference: U.S. Pat. Nos. 6,699,975; 6,790,945;and 8,114,979.

Embodiments Employing Two or Three Optical Labels

In some embodiments, as few as two different kinds of nucleotide arelabeled with different optical labels that generate distinguishableoptical signals for the selected kinds of nucleotide in both sensestrands and antisense strands of target polynucleotides. For example,C's and T's of the complementary strands of each target polynucleotidemay be replaced by labeled analogs, wherein the labels of the C and Tanalogs are capable of generating distinct optical signals. Opticalsignatures are then generated by translocating the labeled strandsthrough nanopores where nucleotides of the strands are constrained topass sequentially through an optical detection region where their labelsare caused to generate optical signals. In some embodiments, informationfrom optical signatures from both sense and antisense strands arecombined to determine a nucleotide sequence of target polynucleotides.

In some embodiments, the selected kinds of nucleotides of targetpolynucleotides are replaced by labeled nucleotide analogs in anextension reaction using a nucleic acid polymerase. Labeled strands oftarget polynucleotides are translocated through nanopores that constrainthe nucleotides of strands to move single file through an opticaldetection region where they are excited so that they produce an opticalsignal. A collection of optical signals for an individual strand isreferred to herein as an optical signature of the strand. In someembodiments, where a strand and its complement (i.e. sense and antisensestrands) are linked, for example, via a hairpin adaptor, a singleoptical signature may include optical signals from optical labels onnucleotides from both the sense strand and the antisense strand. Inother embodiments, different strands of a target polynucleotide mayseparately generate two different optical signatures which may becombined, or used together, for analysis, as mentioned above. Suchseparately analyzed strands may be associated after generation ofoptical signatures, for example, by using molecular tags (which may be,for example, oligonucleotide segments attached to target polynucleotidesin a known position, length and sequence pattern and diversity to permitready association). As noted below, optical signature of the inventionmay comprise mixed optical signals in that the signal detected in eachdetection interval may comprise contributions from multiple opticallabels emitting within a resolution limited area or volume; that is,they may (for example) be mixed FRET signals, as described by Huber etal, U.S. patent publication US20160076091, which is incorporated hereinby reference.

As mentioned above, in some embodiments, methods of the invention may beimplemented with the following steps: (a) copying a strand of a doublestranded polynucleotide so that nucleotide analogs with distinct opticallabels are substituted for at least two kinds of nucleotide to form alabeled strand; (b) copying a complement of the strand so that saidnucleotide analogs are substituted for the same at least two kinds ofnucleotide to form a labeled complement; (c) translocating the labeledstand through a nanopore so that the nucleotides of the labeled strandpass single file through a detection zone where optical labels areexcited to generate optical signals; (d) quenching fluorescent signalsfrom excited fluorescent labels outside of the detection zone; (e)detecting a time series of optical signals from the optical labels asthe labeled strand translocates through the nanopore to produce a strandoptical signature; (f) translocating the labeled complement through ananopore so that the nucleotides of the labeled complement pass singlefile through an excitation zone where optical labels are excited togenerate optical signals; (g) quenching fluorescent signals from excitedfluorescent labels outside of the detection zone; (h) detecting a timeseries of optical signals from the optical labels as the labeledcomplement translocates through the nanopore to produce a complementoptical signature; (i) determining a sequence of the double strandedpolynucleotide from the strand optical signature and the complementoptical signature. In some embodiments, two kinds of nucleotide arelabeled, which may be C's and T's, C's and G's, C's and A's, T's andG's, T's and A's, or G's and A's. In some embodiments, pyrimidinenucleotides are labeled. In other embodiments, purine nucleotides arelabeled. In some embodiments, selected kinds of nucleotides of a strandare labeled by incorporating labeled analog dNTPs of the selected kindof nucleotides in a primer extension reaction using a nucleic acidpolymerase. In other embodiments, selected kinds of nucleotides of astrand are labeled by incorporating analog dNTPs of the selected kindsof nucleotides in an extension reaction, wherein the analog dNTPs arederivatized with orthogonally reactive functionalities that allowattachment of different labels to different kinds of nucleotides in asubsequent reaction. This latter labeling approach is disclosed in Jettet al, U.S. Pat. No. 5,405,747, which is incorporated herein byreference.

In some embodiments, three kinds of nucleotide are labeled, which mayinclude labeling C's with a first optical label, T's with a secondoptical label, and G's and A's with a third optical label. In otherembodiments, the following groups of nucleotides may be labeled asindicated: C's and G's with a first optical label and second opticallabel, respectively, and T's and A's with a third optical label; C's andA's with a first optical label and second optical label, respectively,and T's and G's with a third optical label; T's and G's with a firstoptical label and second optical label, respectively, and C's and A'swith a third optical label; A's and G's with a first optical label andsecond optical label, respectively, and T's and C's with a third opticallabel.

In some embodiments, optical labels are fluorescent acceptor moleculesthat generate a fluorescent resonance energy transfer (FRET) signalafter energy transfer from a donor associated with a nanopore. In someembodiments, as described further below, donors may be optically activenanoparticles, such as, quantum dots, nanodiamonds, or the like.Selection of particular combinations of acceptor molecules and donorsare design choices for one of ordinary skill in the art. In someembodiments, some of which are described more fully below, a singlequantum dot is attached to a nanopore and is excited to fluoresce usingan excitation beam whose wavelength is sufficiently separated, usuallylower (i.e. bluer), so that it does not contribute to FRET signalsgenerated by acceptors. Likewise, a quantum dot is selected whoseemission wavelength overlaps the absorption bands of both acceptormolecules to facilitate FRET interactions. In some embodiments, twodonors may be used for each excitation zone of a nanopore, wherein theemission wavelength of each is selected to optimally overlap theabsorption band of a different one of the acceptor molecules.

In FIG. 6A, double stranded target polynucleotide (600) (SEQ ID NO: 1)consists of sense strand (601) and complementary antisense strand (602),to which is ligated (603) “Y” adaptors (604) and (606) usingconventional methods, e.g. Weissman et al, U.S. Pat. No. 6,287,825;Schmitt et al, U.S. patent publication US2015/004468; which areincorporated herein by reference. Arms (608) and (610) of adaptors (604and 606, respectively) include primer binding sites to which primers(616) and (618) are annealed (605). Double stranded portions (612) and(614) may include tag sequences, e.g. one or both may include randomersof predetermined length and composition, which may be used for laterre-association of the strands, for example, to obtain sequenceinformation from the respective optical signatures of the strands. Afterannealing primers (616) and (618), they may be extended (607) by anucleic acid polymerase in the presence of (for example, as illustrated)labeled dUTP analogs (labels shown as open circles in the incorporatednucleotides) and labeled dCTP analogs (labels shown as filled circles inthe incorporated nucleotides) and natural unlabeled dGTPs and dATPs(with neither unlabeled dTTP nor unlabeled dCTP being present so thatthe analogs are fully substituted in the extended strands). The absenceof labels on G's and A's are illustrated as dashes above theincorporated nucleotides. In an ideal detection system without noise,the sequence of open circles, filled circles and dashes would be goodrepresentations of optical signatures generated by the indicated senseand antisense strands as they pass through an excitation zone of ananopore.

In FIG. 6B, extension products (620) and (622) are illustrated for analternative embodiment employing three labels. Incorporated labeled dUTPanalogs are shown as open circles and incorporated labeled dCTP analogsare shown as filled circles, as above. Incorporated labeled dATP anddGTP analogs are shown as filled diamonds.

Guidance in selecting the kinds of nucleotide to label, kinds of labelsand linkers for attaching them to bases, and nucleic acid polymerasesfor extension reactions in the presence of dNTP analogs can be found inthe following references, which are incorporated by reference: Goodmanet al, U.S. Pat. No. 5,945,312; Jett et al, U.S. Pat. No. 5,405,747;Muehlegger et al, U.S. patent publication US2004/0214221; Giller et al,Nucleic Acids Research, 31(10): 2630-2635 (2003); Tasara et al, NucleicAcids Research, 31(10): 2636-2646 (2003); Augustin et al, J.Biotechnology, 86: 289-301 (2001); Brakmann, Current PharmacueticalBiotechnology, 5(1): 119-126 (2004); and the like. Exemplary nucleicacid polymerases for use with the invention include, but are not limitedto, Vent exo⁻, Taq, E. coli Pol I, Tgo exo⁻, Klenow fragment exo⁻, DeepVent exo⁻, and the like. In some embodiments, exemplary nucleic acidpolymerases include, but are not limited to, Vent exo⁻ and Klenowfragment exo⁻. Exemplary fluorescent labels for dNTP analogs include,but are not limited to, Alexa 488, AMCA, Atto 655, Cy3, Cy5, Evoblue 30,fluorescein, Gnothis blue 1, Gnothis blue 2, Gnothis blue 3, Dy630,Dy635, MR121, rhodamine, Rhodamine Green, Oregon Green, TAMRA, and thelike. Exemplary fluorescent labels for dUTP analogs include, but are notlimited to, Alexa 488, AMCA, Atto 655, Cy3, Cy5, Dy630, Dy665, Evoblue30, Evoblue 90, fluorescein, Gnothis blue 1, Gnothis blue 2, Gnothisblue 3, MR121, Oregon Green, rhodamine, Rhodamine Green, TAMRA, and thelike. Exemplary fluorescent labels for dCTP analogs include, but are notlimited to, Atto 655, Cy5, Evoblue 30, Gnothis blue 3, rhodamine,Rhodamine Green, TAMRA, and the like. Exemplary fluorescent labels fordATP analogs include, but are not limited to, Atto 655, Cy5, Evoblue 30,Gnothis blue 3, Rhodamine Green, and the like. Exemplary fluorescentlabels for dGTP analogs include, but are not limited to, Evoblue 30,Gnothis blue 3, Rhodamine Green, and the like. Exemplary pairs offluorescent labels for dUTP analogs and dCTP analogs include, but arenot limited to, (TAMRA, Rhodamine Green), (Atto 655, Evoblue 30),(Evoblue 30, Atto 655), (Evoblue 30, Gnothis blue 3), (Evoblue 30,Rhodamine Green), (Gnothis blue 1, Rhodamine Green), (Gnothis blue 2,Atto 655), Gnothis blue 3, Cy5), and the like.

FIG. 6C illustrates an embodiment in which two labels are used and senseand antisense strands are linked by means of hairpin adaptor (630), forexample, as taught in U.S. patent publications US 2015/0152492 and US2012/0058468, which are incorporated herein by reference. Tailed adaptor(632) and hairpin adaptor (630) are ligated to target polynucleotide(600) (SEQ ID NO: 1). After denaturation and annealing of primer (634),an extension reaction produces extension product (635) which includessegment (636), the labeled complement of strand (601) and segment (638),the labeled reverse complement of strand (601). After translocation ofextension product (635) through a nanopore and generation of an opticalsignature the sequence of target polynucleotide (600) (SEQ ID NO: 1) canbe determined. Optionally, the sequence of hairpin (630) may be selectedso that a predetermined pattern of labels is incorporated during theextension reaction, which may be used to assist in the analysis of theoptical signature, e.g. by indicating where segment (636) ends and wheresegment (638) begins, or the like.

Nanopores and Nanopore Arrays

Nanopores used with the invention may be solid-state nanopores, proteinnanopores, or hybrid nanopores comprising protein nanopores or organicnanotubes such as carbon or graphene nanotubes, configured in asolid-state membrane, or like framework. Important features of nanoporesinclude constraining polynucleotide analytes, such as labeledpolynucleotides so that their monomers pass through a signal generationregion (or equivalently, an excitation zone, or detection zone, or thelike) in sequence. That is, a nanopore constrains the movement of apolynucleotide analyte, such as a polynucleotide, so that nucleotidespass through a detection zone (or excitation region) in single file. Insome embodiments, additional functions of nanopores include (i) passingsingle stranded nucleic acids while not passing double stranded nucleicacids, or equivalently bulky molecules and/or (ii) constrainingfluorescent labels on nucleotides so that fluorescent signal generationis suppressed or directed so that it is not collected.

In some embodiments, nanopores used in connection with the methods anddevices of the invention are provided in the form of arrays, such as anarray of clusters of nanopores, which may be disposed regularly on aplanar surface. In some embodiments, clusters are each in a separateresolution limited area so that optical signals from nanopores ofdifferent clusters are distinguishable by the optical detection systememployed, but optical signals from nanopores within the same clustercannot necessarily be assigned to a specific nanopore within suchcluster by the optical detection system employed.

Solid state nanopores may be fabricated in a variety of materialsincluding but not limited to, silicon nitride (Si₃N₄), silicon dioxide(SiO₂), and the like. The fabrication and operation of nanopores foranalytical applications, such as DNA sequencing, are disclosed in thefollowing exemplary references that are incorporated by reference: Ling,U.S. Pat. No. 7,678,562; Hu et al, U.S. Pat. No. 7,397,232; Golovchenkoet al, U.S. Pat. No. 6,464,842; Chu et al, U.S. Pat. No. 5,798,042;Sauer et al, U.S. Pat. No. 7,001,792; Su et al, U.S. Pat. No. 7,744,816;Church et al, U.S. Pat. No. 5,795,782; Bayley et al, U.S. Pat. No.6,426,231; Akeson et al, U.S. Pat. No. 7,189,503; Bayley et al, U.S.Pat. No. 6,916,665; Akeson et al, U.S. Pat. No. 6,267,872; Meller et al,U.S. patent publication 2009/0029477; Howorka et al, Internationalpatent publication WO2009/007743; Brown et al, International patentpublication WO2011/067559; Meller et al, International patentpublication WO2009/020682; Polonsky et al, International patentpublication WO2008/092760; Van der Zaag et al, International patentpublication WO2010/007537; Yan et al, Nano Letters, 5(6): 1129-1134(2005); Iqbal et al, Nature Nanotechnology, 2: 243-248 (2007); Wanunu etal, Nano Letters, 7(6): 1580-1585 (2007); Dekker, Nature Nanotechnology,2: 209-215 (2007); Storm et al, Nature Materials, 2: 537-540 (2003); Wuet al, Electrophoresis, 29(13): 2754-2759 (2008); Nakane et al,Electrophoresis, 23: 2592-2601 (2002); Zhe et al, J. Micromech.Microeng., 17: 304-313 (2007); Henriquez et al, The Analyst, 129:478-482 (2004); Jagtiani et al, J. Micromech. Microeng., 16: 1530-1539(2006); Nakane et al, J. Phys. Condens. Matter, 15 R1365-R1393 (2003);DeBlois et al, Rev. Sci. Instruments, 41(7): 909-916 (1970); Clarke etal, Nature Nanotechnology, 4(4): 265-270 (2009); Bayley et al, U.S.patent publication 2003/0215881; and the like.

In some embodiments, the invention comprises nanopore arrays with one ormore light-blocking layers, that is, one or more opaque layers.Typically nanopore arrays are fabricated in thin sheets of material,such as, silicon, silicon nitride, silicon oxide, aluminum oxide, or thelike, which readily transmit light, particularly at the thicknessesused, e.g. less than 50-100 nm. For electrical detection of analytesthis is not a problem. However, in optically-based detection of labeledmolecules translocating nanopores, light transmitted through an arrayinvariably excites materials outside of intended reaction sites, thusgenerates optical noise, for example, from nonspecific backgroundfluorescence, fluorescence from labels of molecules that have not yetentered a nanopore, or the like. In one aspect, the invention addressesthis problem by providing nanopore arrays with one or morelight-blocking layers that reflect and/or absorb light from anexcitation beam, thereby reducing background noise for optical signalsgenerated at intended reaction sites associated with nanopores of anarray. In some embodiments, this permits optical labels in intendedreaction sites to be excited by direct illumination. In someembodiments, an opaque layer may be a metal layer. Such metal layer maycomprise Sn, Al, V, Ti, Ni, Mo, Ta, W, Au, Ag or Cu. In some embodimentssuch metal layer may comprise Al, Au, Ag or Cu. In still otherembodiments, such metal layer may comprise aluminum or gold, or maycomprise solely aluminum. The thickness of an opaque layer may varywidely and depends on the physical and chemical properties of materialcomposing the layer. In some embodiments, the thickness of an opaquelayer may be at least 5 nm, or at least 10 nm, or at least 40 nm. Inother embodiments, the thickness of an opaque layer may be in the rangeof from 5-100 nm; in other embodiments, the thickness of an opaque layermay be in the range of from 10-80 nm. An opaque layer need not block(i.e. reflect or absorb) 100 percent of the light from an excitationbeam. In some embodiments, an opaque layer may block at least 10 percentof incident light from an excitation beam; in other embodiments, anopaque layer may block at least 50 percent of incident light from anexcitation beam.

Opaque layers or coatings may be fabricated on solid state membranes bya variety of techniques known in the art. Material deposition techniquesmay be used including chemical vapor deposition, electrodeposition,epitaxy, thermal oxidation, physical vapor deposition, includingevaporation and sputtering, casting, and the like. In some embodiments,atomic layer deposition may be used, e.g. U.S. Pat. No. 6,464,842; Weiet al, Small, 6(13): 1406-1414 (2010), which are incorporated byreference.

In some embodiments, a 1-100 nm channel or aperture may be formedthrough a solid substrate, usually a planar substrate, such as amembrane, through which an analyte, such as single stranded DNA, isinduced to translocate. In other embodiments, a 2-50 nm channel oraperture is formed through a substrate; and in still other embodiments,a 2-30 nm, or a 2-20 nm, or a 3-30 nm, or a 3-20 nm, or a 3-10 nmchannel or aperture if formed through a substrate. The solid-stateapproach of generating nanopores offers robustness and durability aswell as the ability to tune the size and shape of the nanopore, theability to fabricate high-density arrays of nanopores on a wafer scale,superior mechanical, chemical and thermal characteristics compared withlipid-based systems, and the possibility of integrating with electronicor optical readout techniques. Biological nanopores on the other handprovide reproducible narrow bores, or lumens, especially in the 1-10nanometer range, as well as techniques for tailoring the physical and/orchemical properties of the nanopore and for directly or indirectlyattaching groups or elements, such as fluorescent labels, which may beFRET donors or acceptors, by conventional protein engineering methods.Protein nanopores typically rely on delicate lipid bilayers formechanical support, and the fabrication of solid-state nanopores withprecise dimensions remains challenging. In some embodiments, solid-statenanopores may be combined with a biological nanopore to form a so-called“hybrid” nanopore that overcomes some of these shortcomings, therebyproviding the precision of a biological pore protein with the stabilityof a solid state nanopore. For optical read out techniques a hybridnanopore provides a precise location of the nanopore which simplifiesthe data acquisition greatly.

In some embodiments, clusters may also be formed by disposing proteinnanopores in lipid bilayers supported by solid phase membrane containingan array of apertures. For example, such an array may comprise aperturesfabricated (e.g. drilled, etched, or the like) in solid phase support.The geometry of such apertures may vary depending on the fabricationtechniques employed. In some embodiments, each such aperture isassociated with, or encompassed by, a separate resolution limited area;however, in other embodiments, multiple apertures may be within the sameresolution limited area. The cross-sectional area of the apertures mayvary widely and may or may not be the same as between differentclusters, although such areas are usually substantially the same as aresult of conventional fabrication approaches. In some embodiments,apertures have a minimal linear dimension (e.g. diameter in the case ofcircular apertures) in the range of from 10 to 200 nm, or have areas inthe range of from about 100 to 3×10⁴ nm². Across the apertures may bedisposed a lipid bilayer. The distribution of protein nanopores peraperture may be varied, for example, by controlling the concentration ofprotein nanopores during inserting step. In such embodiments, clustersof nanopores may comprise a random number of nanopores. In someembodiments, in which protein nanopores insert randomly into apertures,clusters containing one or more apertures on average have a number ofprotein nanopores that is greater than zero; in other embodiments, suchclusters have a number of protein nanopores that is greater than 0.25;in other embodiments, such clusters have a number of protein nanoporesthat is greater than 0.5; in other embodiments, such clusters have anumber of protein nanopores that is greater than 0.75; in otherembodiments, such clusters have a number of protein nanopores that isgreater than 1.0.

In some embodiments, methods and devices of the invention comprise asolid phase membrane, such as a SiN membrane, having an array ofapertures therethrough providing communication between a first chamberand a second chamber (also sometimes referred to as a “cis chamber” anda “trans chamber”) and supporting a lipid bilayer on a surface facingthe second, or trans, chamber. In some embodiments, diameters of theaperture in such a solid phase membrane may be in the range of 10 to 200nm, or in the range of 20 to 100 nm. In some embodiments, such solidphase membranes further include protein nanopores inserted into thelipid bilayer in regions where such bilayer spans the apertures on thesurface facing the trans chamber. In some embodiments, such proteinnanopores are inserted from the cis side of the solid phase membraneusing techniques described herein. In some embodiments, such proteinnanopores have a structure identical to, or similar to, α-hemolysin inthat it comprises a barrel, or bore, along an axis and at one end has a“cap” structure and at the other end has a “stem” structure (using theterminology from Song et al, Science, 274: 1859-1866 (1996)). In someembodiments using such protein nanopores, insertion into the lipidbilayer results in the protein nanopore being oriented so that its capstructure is exposed to the cis chamber and its stem structure isexposed to the trans chamber.

In some embodiments, the present invention may employ hybrid nanoporesin clusters, particularly for optical-based nanopore sequencing ofpolynucleotides. Such nanopores comprise a solid-state orifice, oraperture, into which a protein biosensor, such as a protein nanopore, isstably inserted. A charged polynucleotide may be attached to a proteinnanopore (e.g. alpha hemolysin) by conventional protein engineeringtechniques after which an applied electric field may be used to guide aprotein nanopore into an aperture in a solid-state membrane. In someembodiments, the aperture in the solid-state substrate is selected to beslightly smaller than the protein, thereby preventing it fromtranslocating through the aperture. Instead, the protein will beembedded into the solid-state orifice.

Solid state, or synthetic, nanopores may be prepared in a variety ofways, as exemplified in the references cited above. In some embodimentsa helium ion microscope may be used to drill the synthetic nanopores ina variety of materials, e.g. as disclosed by Yang et al, Nanotechnology,22: 285310 (2011), which is incorporated herein by reference. A chipthat supports one or more regions of a thin-film material, e.g. siliconnitride, that has been processed to be a free-standing membrane isintroduced to the helium ion microscope (HIM) chamber. HIM motorcontrols are used to bring a free-standing membrane into the path of theion beam while the microscope is set for low magnification. Beamparameters including focus and stigmation are adjusted at a regionadjacent to the free-standing membrane, but on the solid substrate. Oncethe parameters have been properly fixed, the chip position is moved suchthat the free-standing membrane region is centered on the ion beam scanregion and the beam is blanked. The HIM field of view is set to adimension (in μm) that is sufficient to contain the entire anticipatednanopore pattern and sufficient to be useful in future optical readout(i.e. dependent on optical magnification, camera resolution, etc.). Theion beam is then rastered once through the entire field of view at apixel dwell time that results in a total ion dose sufficient to removeall or most of the membrane autofluorescence. The field of view is thenset to the proper value (smaller than that used above) to performlithographically-defined milling of either a single nanopore or an arrayof nanopores. The pixel dwell time of the pattern is set to result innanopores of one or more predetermined diameters, determined through theuse of a calibration sample prior to sample processing. This entireprocess is repeated for each desired region on a single chip and/or foreach chip introduced into the HIM chamber.

In some embodiments, a device for implementing the above methods foranalyzing polynucleotides (such as single stranded polynucleotides)typically includes a set of electrodes for establishing an electricfield across the layered membrane and nanopores. Single stranded nucleicacids are exposed to nanopores by placing them in an electrolyte in afirst chamber, which is configured as the “cis” side of the layeredmembrane by placement of a negative electrode in the chamber. Uponapplication of an electric field, the negatively single stranded nucleicacids are captured by nanopores and translocated to a second chamber onthe other side of the layered membrane, which is configured as the“trans” side of membrane by placement of a positive electrode in thechamber. The speed of translocation depends in part on the ionicstrength of the electrolytes in the first and second chambers and theapplied voltage across the nanopores. In optically based detection, atranslocation speed may be selected by preliminary calibrationmeasurements, for example, using predetermined standards of labeledsingle stranded nucleic acids that generate signals at differentexpected rates per nanopore for different voltages. Thus, for DNAsequencing applications, a translocation speed may be selected based onthe signal rates from such calibration measurements. Consequently, fromsuch measurements a voltage may be selected that permits, or maximizes,reliable nucleotide identifications, for example, over an array ofnanopores. In some embodiments, such calibrations may be made usingnucleic acids from the sample of templates being analyzed (instead of,or in addition to, predetermined standard sequences). In someembodiments, such calibrations may be carried out in real time during asequencing run and the applied voltage may be modified in real timebased on such measurements, for example, to maximize the acquisition ofnucleotide-specific signals.

Optical Signal Detection

In some embodiments, an epi-illumination system, in which excitationbeam delivery and optical signal collection occurs through a singleobjective, may be used for direct illumination of labels on a polymeranalyte or donors on nanopores. The basic components of a confocalepi-illumination system for use with the invention is illustrated inFIG. 4. Excitation beam (402) is directed to dichroic (404) and onto(412) objective lens (406) which focuses (410) excitation beam (402)onto layered membrane (400), in which labels are excited directly toemit an optical signal, such as a fluorescent signal, or are excitedindirectly via a FRET interaction to emit an optical signal. Suchoptical signal is collected by objective lens (406) and directed todichroic (404), which is selected so that it passes light of opticalsignal (411) but reflects light of excitation beam (402). Optical signal(411) passes through lens (414) which focuses it through pinhole (416)and onto detector (418). When optical signal (411) comprises fluorescentsignals from multiple fluorescent labels further optical components,filters, beam splitters, monochromators, or the like, may be providedfor further separating the different fluorescent signals from differentfluorescent labels.

In some embodiments, labels on nucleotides may be excited by anevanescence field using an apparatus similar to that shown in FIG. 5,described in Soni et al, Review of Scientific Instruments, 81: 014301(2010); and in U.S. patent publication 2012/0135410, which isincorporated herein by reference. In this apparatus, a very narrowsecond chamber on the trans side of a nanopore or nanopore array permitsan evanescent field to extend from a surface of an underlying glassslide to establish detection zones both at entrances and exits of thenanopores, so that each optical measurement associated with a nanoporecontains contributions from a plurality of labeled nucleotides. Array ofapertures (500) (which may include protein nanopores inserted in a lipidbilayer), may be formed in silicon nitride layer (502), which may have athickness in the range of from 20-100 nm. Silicon nitride layer (502)may be formed on a silicon support layer (503). Second chamber (506) maybe formed by silicon nitride layer (502), silicon dioxide layer (504)which determines the height of second chamber (506), and surface (508)of glass slide (510). Silicon dioxide layer (504) may have a thicknessin the range of from 50-100 nm. A desired evanescent field (507)extending from surface (508) across silicon nitride layer (502) may beestablished by directing light beam (512) at an appropriate anglerelative to glass slide (510) so that TIR occurs. For driving labeledpolynucleotide analytes through array (500), cis(−) conditions may beestablished in first chamber (516) and trans(+) conditions may beestablished in second chamber (506) with electrodes operationallyconnected to first and second chambers (506 and 521).

Definitions

“Evanescent field” means a non-propagating electromagnetic field; thatis, it is an electromagnetic field in which the average value of thePoynting vector is zero.

“FRET” or “Förster, or fluorescence, resonant energy transfer” means anon-radiative dipole-dipole energy transfer mechanism from an exciteddonor fluorophore to an acceptor fluorophore in a ground state. The rateof energy transfer in a FRET interaction depends on the extent ofspectral overlap of the emission spectrum of the donor with theabsorption spectrum of the acceptor, the quantum yield of the donor, therelative orientation of the donor and acceptor transition dipoles, andthe distance between the donor and acceptor molecules, Lakowitz,Principles of Fluorescence Spectroscopy, Third Edition (Springer, 2006).FRET interactions of particular interest are those which result aportion of the energy being transferred to an acceptor, in turn, beingemitted by the acceptor as a photon, with a frequency lower than that ofthe light exciting its donor (i.e. a “FRET signal”). “FRET distance”means a distance between a FRET donor and a FRET acceptor over which aFRET interaction can take place and a detectable FRET signal produced bythe FRET acceptor.

“Kit” refers to any delivery system for delivering materials or reagentsfor carrying out a method of the invention. In the context of reactionassays, such delivery systems include systems that allow for thestorage, transport, or delivery of reaction reagents (e.g., fluorescentlabels, such as mutually quenching fluorescent labels, fluorescent labellinking agents, enzymes, etc. in the appropriate containers) and/orsupporting materials (e.g., buffers, written instructions for performingthe assay etc.) from one location to another. For example, kits includeone or more enclosures (e.g., boxes) containing the relevant reactionreagents and/or supporting materials. Such contents may be delivered tothe intended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a second ormore containers contain mutually quenching fluorescent labels.

“Nanopore” means any opening positioned in a substrate that allows thepassage of analytes through the substrate in a predetermined ordiscernable order, or in the case of polymer analytes, passage of theirmonomeric units through the substrate in a predetermined or discernibleorder. In the latter case, a predetermined or discernible order may bethe primary sequence of monomeric units in the polymer. Examples ofnanopores include proteinaceous or protein based nanopores, synthetic orsolid state nanopores, and hybrid nanopores comprising a solid statenanopore having a protein nanopore embedded therein. A nanopore may havean inner diameter of 1-10 nm or 1-5 nm or 1-3 nm. Examples of proteinnanopores include but are not limited to, alpha-hemolysin,voltage-dependent mitochondrial porin (VDAC), OmpF, OmpC, MspA and LamB(maltoporin), e.g. disclosed in Rhee, M. et al., Trends inBiotechnology, 25(4) (2007): 174-181; Bayley et al (cited above);Gundlach et al, U.S. patent publication 2012/0055792; and the like,which are incorporated herein by reference. Any protein pore that allowsthe translocation of single nucleic acid molecules may be employed. Ananopore protein may be labeled at a specific site on the exterior ofthe pore, or at a specific site on the exterior of one or more monomerunits making up the pore forming protein. Pore proteins are chosen froma group of proteins such as, but not limited to, alpha-hemolysin, MspA,voltage-dependent mitochondrial porin (VDAC), Anthrax porin, OmpF, OmpCand LamB (maltoporin). Integration of the pore protein into the solidstate hole is accomplished by attaching a charged polymer to the poreprotein. After applying an electric field the charged complex iselectrophoretically pulled into the solid state hole. A syntheticnanopore, or solid-state nanopore, may be created in various forms ofsolid substrates, examples of which include but are not limited tosilicones (e.g. Si3N4, SiO2), metals, metal oxides (e.g. Al2O3)plastics, glass, semiconductor material, and combinations thereof. Asynthetic nanopore may be more stable than a biological protein porepositioned in a lipid bilayer membrane. A synthetic nanopore may also becreated by using a carbon nanotube embedded in a suitable substrate suchas but not limited to polymerized epoxy. Carbon nanotubes can haveuniform and well-defined chemical and structural properties. Varioussized carbon nanotubes can be obtained, ranging from one to hundreds ofnanometers. The surface charge of a carbon nanotube is known to be aboutzero, and as a result, electrophoretic transport of a nucleic acidthrough the nanopore becomes simple and predictable (Ito, T. et al.,Chem. Commun. 12 (2003): 1482-83). The substrate surface of a syntheticnanopore may be chemically modified to allow for covalent attachment ofthe protein pore or to render the surface properties suitable foroptical nanopore sequencing. Such surface modifications can be covalentor non-covalent. Most covalent modification include an organosilanedeposition for which the most common protocols are described: 1)Deposition from aqueous alcohol. This is the most facile method forpreparing silylated surfaces. A 95% ethanol-5% water solution isadjusted to pH 4.5-5.5 with acetic acid. Silane is added with stirringto yield a 2% final concentration. After hydrolysis and silanol groupformation the substrate is added for 2-5 min. After rinsed free ofexcess materials by dipping briefly in ethanol. Cure of the silane layeris for 5-10 min at 110 degrees Celsius. 2) Vapor Phase Deposition.Silanes can be applied to substrates under dry aprotic conditions bychemical vapor deposition methods. These methods favor monolayerdeposition. In closed chamber designs, substrates are heated tosufficient temperature to achieve 5 mm vapor pressure. Alternatively,vacuum can be applied until silane evaporation is observed. 3) Spin-ondeposition. Spin-on applications can be made under hydrolytic conditionswhich favor maximum functionalization and polylayer deposition or dryconditions which favor monolayer deposition. In some embodiments, singlenanopores are employed with methods of the invention. In otherembodiments, a plurality of nanopores are employed. In some of thelatter embodiments, a plurality of nanopores is employed as an array ofnanopores, usually disposed in a planar substrate, such as a solid phasemembrane. Nanopores of a nanopore array may be spaced regularly, forexample, in a rectilinear pattern, or may be spaced randomly. In apreferred embodiment, nanopores are spaced regularly in a rectilinearpattern in a planar solid phase substrate.

“Polymer” means a plurality of monomers connected into a linear chain.Usually, polymers comprise more than one type of monomer, for example,as a polynucleotide comprising A's, C's, G's and T's, or a polypeptidecomprising more than one kind of amino acid. Monomers may includewithout limitation nucleosides and derivatives or analogs thereof andamino acids and derivatives and analogs thereof. In some embodiments,polymers are polynucleotides, whereby nucleoside monomers are connectedby phosphodiester linkages, or analogs thereof.

“Polynucleotide” or “oligonucleotide” are used interchangeably and eachmean a linear polymer of nucleotide monomers or analogs thereof.Monomers making up polynucleotides and oligonucleotides are capable ofspecifically binding to a natural polynucleotide by way of a regularpattern of monomer-to-monomer interactions, such as Watson-Crick type ofbase pairing, base stacking, Hoogsteen or reverse Hoogsteen types ofbase pairing, or the like. Such monomers and their internucleosidiclinkages may be naturally occurring or may be analogs thereof, e.g.naturally occurring or non-naturally occurring analogs. Non-naturallyoccurring analogs may include PNAs, phosphorothioate internucleosidiclinkages, bases containing linking groups permitting the attachment oflabels, such as fluorophores, or haptens, and the like. Whenever the useof an oligonucleotide or polynucleotide requires enzymatic processing,such as extension by a polymerase, ligation by a ligase, or the like,one of ordinary skill would understand that oligonucleotides orpolynucleotides in those instances would not contain certain analogs ofinternucleosidic linkages, sugar moieties, or bases at any or somepositions. Polynucleotides typically range in size from a few monomericunits, e.g. 5-40, when they are usually referred to as“oligonucleotides,” to several thousand monomeric units. Whenever apolynucleotide or oligonucleotide is represented by a sequence ofletters (upper or lower case), such as “ATGCCTG,” it will be understoodthat the nucleotides are in 5′3′ order from left to right and that “A”denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotesdeoxyguanosine, and “T” denotes thymidine, “I” denotes deoxyinosine, “U”denotes uridine, unless otherwise indicated or obvious from context.Unless otherwise noted the terminology and atom numbering conventionswill follow those disclosed in Strachan and Read, Human MolecularGenetics 2 (Wiley-Liss, New York, 1999). Usually polynucleotidescomprise the four natural nucleosides (e.g. deoxyadenosine,deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribosecounterparts for RNA) linked by phosphodiester linkages; however, theymay also comprise non-natural nucleotide analogs, e.g. includingmodified bases, sugars, or internucleosidic linkages. It is clear tothose skilled in the art that where an enzyme has specificoligonucleotide or polynucleotide substrate requirements for activity,e.g. single stranded DNA, RNA/DNA duplex, or the like, then selection ofappropriate composition for the oligonucleotide or polynucleotidesubstrates is well within the knowledge of one of ordinary skill,especially with guidance from treatises, such as Sambrook et al,Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, NewYork, 1989), and like references. Likewise, the oligonucleotide andpolynucleotide may refer to either a single stranded form or a doublestranded form (i.e. duplexes of an oligonucleotide or polynucleotide andits respective complement). It will be clear to one of ordinary skillwhich form or whether both forms are intended from the context of theterms usage.

“Primer” means an oligonucleotide, either natural or synthetic that iscapable, upon forming a duplex with a polynucleotide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3′ end along the template so that an extended duplex is formed.Extension of a primer is usually carried out with a nucleic acidpolymerase, such as a DNA or RNA polymerase. The sequence of nucleotidesadded in the extension process is determined by the sequence of thetemplate polynucleotide. Usually primers are extended by a DNApolymerase. Primers usually have a length in the range of from 14 to 40nucleotides, or in the range of from 18 to 36 nucleotides. Primers areemployed in a variety of nucleic amplification reactions, for example,linear amplification reactions using a single primer, or polymerasechain reactions, employing two or more primers. Guidance for selectingthe lengths and sequences of primers for particular applications is wellknown to those of ordinary skill in the art, as evidenced by thefollowing references that are incorporated by reference: Dieffenbach,editor, PCR Primer: A Laboratory Manual, 2nd Edition (Cold Spring HarborPress, New York, 2003).

“Resolution limited area” is an area of a surface of a nanopore ornanowell array within which individual features or light emissionsources cannot be distinguished by an optical signal detection system.Without intending to be limited by theory, such resolution limited areais determined by a resolution limit (also sometimes referred to as a“diffraction limit” or “diffraction barrier”) of an optical system. Suchlimit is determined by the wavelength of the emission source and theoptical components and may be defined by d=λ/NA, where d is the smallestfeature that can be resolved, λ is the wavelength of the light and NA isthe numerical aperture of the objective lens used to focus the light.Thus, whenever two or more nanopores are within a resolution limitedarea and two or more optical signals are generated at the respectivenanopores, an optical detection system cannot distinguish or determinewhich optical signals came from which nanopore. In accordance with theinvention, a surface of a nanopore array may be partitioned, orsubdivided, into non-overlapping regions, or substantiallynon-overlapping regions, corresponding to resolution limited areas. Thesize of such subdivisions corresponding to resolution limited areas maydepend on a particular optical detection system employed. In someembodiments, whenever light emission sources are within the visiblespectrum, a resolution limited area is in the range of from 300 nm² to3.0 μm²; in; other embodiments, a resolution limited area is in therange of from 1200 nm² to 0.7 μm²; in other embodiments, a resolutionlimited area is in the range of from 3×10⁴ nm² to 0.7 μm², wherein theforegoing ranges of areas are in reference to a surface of a nanopore ornanowell array. In some embodiments, the visible spectrum meanswavelengths in the range of from about 380 nm to about 700 nm.

“Sequence determination”, “sequencing” or “determining a nucleotidesequence” or like terms in reference to polynucleotides includesdetermination of partial as well as full sequence information of thepolynucleotide. That is, the terms include sequences of subsets of thefull set of four natural nucleotides, A, C, G and T, such as, forexample, a sequence of just A's and C's of a target polynucleotide. Thatis, the terms include the determination of the identities, ordering, andlocations of one, two, three or all of the four types of nucleotideswithin a target polynucleotide. In some embodiments, the terms includethe determination of the identities, ordering, and locations of two,three or all of the four types of nucleotides within a targetpolynucleotide. In some embodiments sequence determination may beaccomplished by identifying the ordering and locations of a single typeof nucleotide, e.g. cytosines, within the target polynucleotide “catcgc. . . ” so that its sequence is represented as a binary code, e.g.“100101 . . . ” representing “c-(not c)(not c)c-(not c)-c . . . ” andthe like. In some embodiments, the terms may also include subsequencesof a target polynucleotide that serve as a fingerprint for the targetpolynucleotide; that is, subsequences that uniquely identify a targetpolynucleotide, or a class of target polynucleotides, within a set ofpolynucleotides, e.g. all different RNA sequences expressed by a cell.

This disclosure is not intended to be limited to the scope of theparticular forms set forth, but is intended to cover alternatives,modifications, and equivalents of the variations described herein.Further, the scope of the disclosure fully encompasses other variationsthat may become obvious to those skilled in the art in view of thisdisclosure. The scope of the present invention is limited only by theappended claims.

What is claimed is:
 1. A method for determining a nucleotide sequence ofa polynucleotide comprising the steps of: translocating a polynucleotidethrough a bore of a nanopore at a translocation speed, whereinnucleotides of the polynucleotide are labeled with fluorescent labelssuch that, when exposed to fluorescence excitation light, fluorescentlabels of nucleotides that are in free solution are substantiallyquenched, and fluorescent labels of nucleotides within the bore areconstrained such that substantially no detectable fluorescent signal isgenerated by the fluorescent labels therein; exciting the fluorescentlabel of each nucleotide upon exiting the nanopore and prior to beingquenched by a preceding mutually quenching fluorescent label of thepolynucleotide or by a quenching agent in the solution; measuring afluorescent signal generated by fluorescent labels of the nucleotidesexiting the nanopore, wherein the translocation speed is selected sothat a fluorescent label of an exiting nucleotide is quenched before ittravels more than an inter-nucleotide distance from the nanopore; anddetermining a nucleotide sequence of the polynucleotide from a sequenceof measured fluorescent signals.
 2. The method of claim 1 wherein saidstep of measuring includes selecting said translocation speed based onthe relative values of fluorescent intensities distributed among aplurality of optical detection channels.
 3. The method of claim 2wherein said plurality of optical detection channels includes at leastone channel for an emission maximum of each fluorescent label.
 4. Amethod of determining a nucleotide sequences of polynucleotides, themethod comprising the steps of: (a) translocating single strandedpolynucleotides through nanopores at a translocation speed, whereinnucleotides of the single stranded polynucleotides are labeled withfluorescent labels such that, when exposed to fluorescence excitationlight, fluorescent labels of adjacent nucleotides are in a mutuallyquenched state in free solution and wherein the nanopores force thefluorescent labels within the nanopore into a constrained state whereinsubstantially no detectable signal is generated; (b) exciting thefluorescent labels of nucleotides upon exiting the nanopores and priorto being quenched by the label of an adjacent nucleotide; (c) measuringfluorescent signals generated by the fluorescent labels exiting thenanopores and determining from the measured fluorescent signals whethera single fluorescent label contributes to the measured fluorescentsignal or a plurality of fluorescent labels contributes to the measuredfluorescent signal at each nanopore; (d) reducing the translocationspeed by a predetermined value whenever a measured fluorescent signal ata nanopore is generated by a plurality of fluorescent labels; (e)repeating steps (a) through (d) until the translocation speedcorresponds to substantially every measured fluorescent signal at eachnanopore comprising fluorescence from substantially a single fluorescentlabel; and (f) identifying nucleotides of the translocatingpolynucleotides from measured fluorescent signals.
 5. The method ofclaim 4 wherein said step of reducing comprises lowering a voltagepotential across said nanopores.
 6. The method of claim 4 whereinnucleotides of said polynucleotides are labeled with second members of aFRET pair, each second member producing a FRET signal indicative of thenucleotide to which it is attached, and wherein nucleotides of saidpolynucleotides pass in sequence by a first member of the FRET pairpositioned adjacent to said nanopore so that each second member uponexiting said nanopore passes within a FRET distance of the first memberof the FRET pair; and wherein said step of exciting includes exposingthe first member to excitation energy of a first wavelength so that FREToccurs between the first and second members of the FRET pair within theFRET distance to generate a FRET signal of a second wavelengthindicative of the nucleotide exiting said nanopore.
 7. The method ofclaim 6 wherein said nanopore is disposed in a solid phase membrane andwherein said first member of said FRET pair is attached to the solidphase membrane adjacent to said nanopore.
 8. The method of claim 6wherein said nanopore is a protein nanopore and wherein said firstmember of said FRET pair is attached to the protein nanopore.
 9. Themethod of claim 6 wherein said first member of said FRET pair is a donorand said second member of said FRET pair is an acceptor.
 10. The methodof claim 9 wherein said donor is a quantum dot.
 11. The method of claim9 wherein said acceptor is a fluorescent organic dye.